Macropinocytosis has emerged as an important pathway of protein acquisition in cancer cells, particularly in tumors with activated Ras such as pancreatic and colon cancer. Macropinocytosis is also the route of entry of Bacillus Calmette-Guerin (BCG) and other microbial therapies of cancer. Despite this important role in tumor biology and therapy, the full mechanisms by which cancer cells can activate macropinocytosis remain incompletely defined. Using BCG uptake to assay macropinocytosis, we executed a genome-wide shRNA screen for macropinocytosis activators and identified Wnt pathway activation as a strong driver of macropinocytosis. Wnt-driven macropinocytosis was downstream of the β-catenin-dependent canonical Wnt pathway, was PAK1 dependent, and supported albumin-dependent growth in Ras-WT cells. In cells with activated Ras-dependent macropinocytosis, pharmacologic or genetic inhibition of Wnt signaling suppressed macropinocytosis. In a mouse model of Wnt-driven colonic hyperplasia via silencing, Wnt-activated macropinocytosis stimulated uptake of luminal microbiota, a process reversed by topical pharmacologic inhibition of macropinocytosis. Our findings indicate that Wnt pathway activation drives macropinocytosis in cancer, and its inhibition could provide a therapeutic vulnerability in Wnt-driven intestinal polyposis and cancers with Wnt activation. The Wnt pathway drives macropinocytosis in cancer cells, thereby contributing to cancer growth in nutrient-deficient conditions and, in the context of colon cancer, to the early phases of oncogenesis. .

Bacillus Calmette–Guérin (BCG) immunotherapy for bladder cancer is the only bacterial cancer therapy approved for clinical use. Although presumed to induce T cell-mediated immunity, whether tumor elimination depends on bacteria-specific or tumor-specific immunity is unknown. Herein we show that BCG-induced bladder tumor elimination requires CD4 and CD8 T cells, although augmentation or inhibition of bacterial antigen-specific T cell responses does not alter the efficacy of BCG-induced tumor elimination. In contrast, BCG stimulates long-term tumor-specific immunity that primarily depends on CD4 T cells. We demonstrate that BCG therapy results in enhanced effector function of tumor-specific CD4 T cells, mainly through enhanced production of IFN-γ. Accordingly, BCG-induced tumor elimination and tumor-specific immune memory require tumor cell expression of the IFN-γ receptor, but not MHC class II. Our findings establish that a bacterial immunotherapy for cancer is capable of inducing tumor immunity, an antitumor effect that results from enhanced function of tumor-specific CD4 T cells, and ultimately requires tumor-intrinsic IFN-γ signaling, via a mechanism that is distinct from other tumor immunotherapies.

For decades, BCG immunotherapy has been the standard of care for non–muscle-invasive bladder cancer. Despite this clinical experience, the mechanism by which BCG stimulates tumor-eliminating immunity is unclear, and there is still a need for more accurate prediction of clinical outcomes in advance of treatment initiation. We have shown that BCG stimulates tumor-specific T-cell immunity that requires tumor cell expression of the IFNγ receptor (IFNGR); however, the downstream components of IFNGR signaling responsible for responsiveness to BCG are unknown. Here, we demonstrate that the IFNγ-driven, tumor cell intrinsic expression of the class II transactivator CIITA is required for activation of a tumor-specific CD4 T-cell response and BCG-induced tumor immunity. Despite the established role for CIITA in controlling MHC-II antigen presentation machinery, the requirement for CIITA is independent of MHC-II and associated genes. Rather, we find that CIITA is required for a broader tumor-intrinsic transcriptional program linked to critical pathways of tumor immunity via mechanisms that remain to be determined. Tumor cell intrinsic expression of CIITA is not required for a response to immunotherapy targeting programmed cell death protein 1 (PD-1), suggesting that different modalities of immunotherapy for bladder cancer could be employed based on tumor-intrinsic characteristics.

<p>Table S1: A Whole-genome shRNA screen for inhibitors of macropinocytosis</p>

<p>Table S2: A Whole-genome shRNA screen for inhibitors of macropinocytosis</p>